HCMV Displays a Unique Transcriptome of Immunomodulatory Genes in Primary Monocyte-Derived Cell Types

<div><p>Human cytomegalovirus (HCMV) is a betaherpesvirus which rarely presents problems in healthy individuals, yet may result in severe morbidity in immunocompromised patients and in immune-naïve neonates. HCMV has a large 235 kb genome with a coding capacity of at least 165 open reading frames (ORFs). This large genome allows complex gene regulation resulting in different sets of transcripts during lytic and latent infection. While latent virus mainly resides within monocytes and CD34⁺ progenitor cells, reactivation to lytic infection is driven by differentiation towards terminally differentiated myeloid dendritic cells and macrophages. Consequently, it has been suggested that macrophages and dendritic cells contribute to viral spread <i>in vivo</i>. Thus far only limited knowledge is available on the expression of HCMV genes in terminally differentiated myeloid primary cells and whether or not the virus exhibits a different set of lytic genes in primary cells compared with lytic infection in NHDF fibroblasts. To address these questions, we used Illumina next generation sequencing to determine the HCMV transcriptome in macrophages and dendritic cells during lytic infection and compared it to the transcriptome in NHDF fibroblasts. Here, we demonstrate unique expression profiles in macrophages and dendritic cells which significantly differ from the transcriptome in fibroblasts mainly by modulating the expression of viral transcripts involved in immune modulation, cell tropism and viral spread. In a head to head comparison between macrophages and dendritic cells, we observed that factors involved in viral spread and virion composition are differentially regulated suggesting that the plasticity of the virion facilitates the infection of surrounding cells. Taken together, this study provides the full transcript expression analysis of lytic HCMV genes in monocyte-derived type 1 and type 2 macrophages as well as in monocyte-derived dendritic cells. Thereby underlining the potential of HCMV to adapt to or influence different cellular environments to promote its own survival.</p></div

Percentage of mapped reads to the HCMV transcriptome in primary cell types.

<p>Six different blood donors (A-F) were processed to obtain monocytes which were differentiated to DCs, MΦ1 and MΦ2. Subsequently, these primary cell types were infected with TB40/E at MOI5. As a comparison, we infected NHDF cells (n = 3) at MOI 0.16, MOI 0.5 and MOI 5. Given in the top panel are the percentages of mapped reads on the HCMV genome for each cell type.</p

Enrichment of functional groups when comparing DCs with both types of macrophages and when comparing MΦ1 with MΦ2 macrophages.

<p>The HCMV was annotated and a mathematical model was used to determine which functional groups were significantly enriched in the differentially regulated genes. Based on this algorithm, genes involved in cell tropism, viral spread and immunomodulation are the most enriched groups in DCs, MΦ2 and MΦ1.</p

Expression levels of the HCMV genes transcriptome in fibroblasts (MOI 0.16).

<p>Log(10) of the RPKM values per gene were binned in six categories and mapped on the reference genome of TB40/E.</p

Differential regulation of HCMV gene expression in monocyte-derived DCs, MΦ1 and MΦ2 (MOI5) compared to lytic infection in NHDF fibroblasts (MOI0.16).

<p>On the graph is the fold increase or decrease (reflecting genes which are expressed higher/lower in fibroblasts than in primary cell types). Red bars represent significant changes in gene expression between the indicated cell type and NHDF fibroblasts (adjusted p<0.05), black bars indicate modulated genes which did not reach the significance threshold (adjusted p≥0.05).</p

Enrichment of functional groups in DCs, MΦ1 and MΦ2 compared to NHDF.

<p>Genes involved in cell tropism, viral spread and immunomodulation are shown to be the most enriched groups in DCs, MΦ2 and MΦ1. Given are the p-vales of each individual gene.</p